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The aim of this study was to monitor seven phosphatidylethanol (PEth) homologues in dried blood spots (DBS) and ethyl glucuronide in hair (EtGH) over a 6-month period of drinking while documenting the daily drinks (amount and type) of alcohol via app. A total of 23 volunteers (12 males and 11 females) aged 19-54 years were enrolled. At four-weekly intervals, capillary blood to create DBS and after 3 and 6 months, respectively, a strand of hair (proximal, 3 cm) was collected. Analyses of EtGH and PEth homologues were performed using liquid chromatography-tandem mass spectrometry. All participants consumed alcohol during the 6 months. Only one participant tested negative for both PEth and EtGH. Eight participants had PEth 16:0/18:1 concentrations between 20 and <210 ng/mL (mean: 45.6 ng/mL) but EtGH concentrations below 5 pg/mg. PEth 16:0/18:1 concentrations between 20 and <210 ng/mL and EtGH concentrations between 5 and <30 pg/mg were assigned to eight subjects, uniformly matching them in the category of socially accepted drinking behavior. Four test subjects exceeded the cutoff for social drinking behavior in both PEth 16:0/18:1 (mean: 528 ng/mL) and EtGH (mean: 84.5 pg/mg). Two participants exceeded the threshold for PEth 16:0/18:1 of 210 ng/mL in blood but remained below 30 pg EtG/mg hair. PEth showed a higher detection rate for alcohol consumption than EtGH did. Moreover, PEth concentrations reacted quickly to changes in drinking behavior, whereas EtGH concentrations remained similar over time., (© 2023 John Wiley & Sons, Ltd.)

Aims: Phosphatidylethanol (PEth) is only formed when ethanol is present in blood. This direct alcohol marker has been widely discussed, including the minimum amount of ethanol being necessary to form as much PEth as to exceed the threshold of 20 ng/mL in previously PEth negative subjects. In order to corroborate hitherto existing results, a drinking study including 18 participants after a 3-week alcohol abstinence was performed., Methods: They consumed a pre-calculated amount of ethanol to reach a blood alcohol concentration (BAC) of at least 0.6 g/kg. Blood was drawn before and periodically seven times after alcohol administration on day 1. Blood and urine were also collected the next morning. Dried blood spots (DBS) were prepared immediately from collected venous blood. BAC was determined by head space gas chromatography and the concentrations of both PEth (16:0/18:1, 16:0/18:2 and five additional homologues) and ethyl glucuronide (EtG) were analysed using liquid chromatography-tandem mass spectrometry., Results: Out of 18, 5 participants had concentrations of PEth 16:0/18:1 above the threshold of 20 ng/mL, and 11 out of the 18 subjects had concentrations between 10 and 20 ng/mL. In addition, four persons had PEth 16:0/18:2 concentrations above 20 ng/mL the following morning. All test subjects tested positive for EtG in DBS (≥ 3 ng/mL) and urine (≥100 ng/mL) upon 20-21 h after alcohol administration., Conclusion: By combining both a lower cutoff of 10 ng/mL and the homologue PEth 16:0/18:2, the sensitivity to detect a single alcohol intake after a 3-week abstinence increases to 72.2%., (© The Author(s) 2023. Medical Council on Alcohol and Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In recent years, numerous new psychoactive substances (NPS) have emerged on the illicit drug market. The assumed non-detectability of these drugs is often a key motivation for individuals subject to drug testing, such as those in driving license regranting programs. In these programs, NPS are not routinely tested for, and thus, subjects who have to prove abstinence from common drugs of abuse might switch to NPS to avoid positive drug tests. The aim of the study was to determine the frequency of these substances in the hair and urine samples of individuals undergoing drug testing in driving license regranting. A total of 1037 samples (577 hair and 460 urine samples) collected from 949 subjects between February 2017 and December 2018 were retrospectively analyzed for designer drugs and synthetic cannabinoids by liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). For a more sensitive analysis of synthetic cannabinoids and their metabolites, additional testing was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, 42 hair and two urine samples, which were obtained from 40 subjects, tested positive for NPS resulting in a frequency of 4.2%. While synthetic cannabinoids were detected in all cases, designer drugs were only found in three of these cases. With regard to the 577 hair samples analyzed, 7.3% screened positive, whereas only 0.4% of the 460 tested urine samples contained NPS. The results of this study indicate that synthetic cannabinoid use seems to be popular among this population, and therefore, testing for synthetic cannabinoids should be requested more often preferably using hair analysis., (© 2023 John Wiley & Sons Ltd.)

Synthetic cannabinoid receptor agonists (SCRAs, "Spice") are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the "Spice"-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs' involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. "Spice"-related fatalities and SCRAs' causative role have significantly decreased among our cases since the German New Psychoactive Substances Act., (© 2023. The Author(s).)

Phosphatidylethanol (PEth) has become a widespread marker offering an up to 4-week retrospective window to detect alcohol use. Due to the pandemic of coronavirus disease 2019, ethanol-based hand sanitizers are frequently used. The aim of this study was to develop and validate a method for the determination of up to seven different homologues of PEth from dried blood spots (DBSs) after use of an ethanol-based hand sanitizer. The objectives of its preliminary application were to prove whether a threshold of 20 ng/mL for PEth 16:0/18:1 is reached and whether other homologues are formed as well as if positive findings of urinary ethyl glucuronide (UEtG) can be observed with respect to assess monitoring of abstinence control programs. Ten volunteers (8 occasional and 2 regular drinkers) were recruited to excessively use an ethanol-based hand sanitizer on 5 successive days. DBSs and urine samples were collected daily. PEth and UEtG were determined by liquid chromatography--tandem mass spectrometry. In total, two volunteers with initial PEth 16:0/18:1 concentrations of 19.3 and 14.6 ng/mL exceeded the threshold of 20 ng/mL six times. Subjects drinking daily or almost daily had starting PEth 16:0/18:1 concentrations of 242 and 354 ng/mL, showing a decline of PEth concentrations in six out of the seven homologues over 5 days. In teetotalers, formation of PEth species could not be observed. Thus, not satisfying requirements in an alcohol monitoring program with initial PEth-negative blood cannot be explained by a frequent use of ethanol-based hand sanitizer only. In cases of regular alcohol consumption, PEth homologues are not likely to be further influenced. However, results indicated that individuals with a PEth concentration close to 20 ng/mL are at risk of exceeding the threshold by using ethanol-based hand sanitizer., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cannabidiol (CBD) products have ascribed an uprising trend for their health-promoting effects worldwide. In contrast to Δ 9 -tetrahydrocannabinol (THC), CBD exhibits no state of euphoria. Since conversion of CBD into THC in an acidic environment has been reported, it has not been proved whether this degradation will also occur in human gastric fluid. A total of 9 subjects ingested 400 mg CBD as a water-soluble liquid together with lecithin as an emulsifier and ethanol as a solubilizer. Blood samples were taken up to 4 h, and urine samples were submitted up to 48 h. THC, 11-hydroxy-Δ 9 -THC (THC-OH), 11-nor-9-carboxy-Δ 9 -THC (THC-COOH), CBD, 7-hydroxy cannabidiol (7-OH-CBD), and 7-carboxy cannabidiol (7-CBD-COOH) were determined in blood and THC-COOH and 7-CBD-COOH in urine by LC-MS/MS. Neither THC, THC-OH, nor THC-COOH were detectable in any serum specimen. Only two urine samples revealed THC-COOH values slightly above the threshold of 10 ng/ml, which could also be caused by trace amounts of THC being present in the CBD liquid. It can be concluded that negative consequences for participants of a drug testing program due to a conversion of CBD into THC in human gastric fluid appear unlikely, especially considering a single intake of dosages of less than 400 mg. Nevertheless, there is a reasonable risk for consumers of CBD products being tested positive for THC or THC metabolites. However, this is probably not caused by CBD cyclization into THC in human gastric fluid but is most likely due to THC being present as an impurity of CBD products., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In hair analysis, identification of 11-nor-9-carboxy-∆ 9 -tetrahydrocannabinol (THC-COOH), one of the major endogenously formed metabolites of the psychoactive cannabinoid tetrahydrocannabinol (THC), is considered unambiguous proof of cannabis consumption. Due to the complex hair matrix and low target concentrations of THC-COOH in hair, this kind of investigation represents a great analytical challenge. The aim of this work was to establish a fast, simple, and reliable LC-MS 3 routine method for sensitive detection of THC-COOH in hair samples. Furthermore, the LC-MS 3 method developed also included the detection of derivatized 11-hydroxy-∆ 9 -THC (11-OH-THC) as an additional marker of cannabis use. Hair sample preparation prior to detection of the two THC metabolites was based on digestion of the hair matrix under alkaline conditions followed by an optimized liquid-liquid extraction (LLE) procedure. Sample preparation by LLE proved to be more suitable than solid-phase extraction (SPE) due to less laborious and time-consuming steps while still yielding satisfactory results. A significant improvement in analytical detection was introduced by multistage fragmentation (MS 3 ), which led to enhanced sensitivity and selectivity and thus low limits of quantification (0.1 pg/mg hair). The MS 3 method included two transitions for THC-COOH (m/z 343 → 299 → 245 and m/z 343 → 299 → 191) encompassing the quantifier (m/z 245) and the qualifier ion (m/z 191). The method was fully validated, and successful application to authentic toxicology case samples was demonstrated by the analysis of more than 2000 hair samples from cannabis users with THC-COOH concentrations determined ranging from 0.1 to >15 pg/mg hair., (© 2022 John Wiley & Sons Ltd.)

A previously published method for single hair analysis has been applied to a doping case for further clarification. Amphetamine could be detected in multiple micro segments resulting in two distinct concentration peaks in several hairs. The consumption of a contaminated food supplement as possible source for the amphetamine is discussed., (© 2022 John Wiley & Sons, Ltd.)

A high volume of fluid can strongly reduce a drug's concentration in urine. Therefore, to detect diluted samples, the concentration of creatinine in urine is determined during testing drugs of abuse. If the concentration is below 20 mg creatinine/dl urine, the urine sample is usually rejected for drug testing. It should be examined whether creatine or creatinine ingestion can mask urine dilution by increasing the creatinine concentration. A total of 18 subjects drank 1.3 L of water and 0.2 L of orange juice on each of the three testing days: (1) without creatine, (2) with 20 g of creatine, and (3) with 20 g of creatine following incubation for 4 days in orange juice at room temperature; an acidic environment should promote conversion of creatine to creatinine. The lowest creatinine concentrations in urine were observed on average 2 h after fluid intake. At that time, ingestion of fluid without creatine, with creatine, and with creatine(ine)-orange juice mixture resulted in mean values of 11.6, 22.5, and 28.3 mg creatinine/dl urine, respectively. It can be concluded that ingestion of creatine or creatinine can increase the concentration of creatinine in urine and thus mask dilution of a sample. The conversion of creatine in orange juice further increases availability of creatinine as it is obvious from urine creatinine concentration. Therefore, creatine ingestion during drug testing will give rise to negative results due to matrix adulteration. In a case of suspected creatine supplementation, the creatine content of the sample should be determined in addition to creatinine., (© 2021 John Wiley & Sons, Ltd.)

Purpose: In forensics, entomological specimens can be used as additional/alternative matrices to detect xenobiotics when human specimens are limited in their application. Despite some advantages over implementing putrefied human remains, most medico-legal laboratories do not include entomotoxicological procedures as routine analytical methods. We thus applied two authentic cases to evaluate necrophagous larvae's potential as complementary matrices for toxicological analysis after extensive postmortem decomposition., Methods: Larvae and postmortem human samples, including hair, stomach contents, pericardial fluid, liver, lung, and skeletal muscle, were collected at autopsy. Samples were analyzed by liquid chromatography-tandem mass spectrometry and liquid chromatography-quadrupole time-of-flight mass spectrometry for pharmaceutical substances, illicit drugs, and new psychoactive substances, including synthetic cannabinoids, benzodiazepines, new synthetic opioids, and stimulants., Results: Nearly all substances detected in human specimens, including several benzodiazepines and synthetic cannabinoids, were also detected in larvae. Surprisingly, some drugs, including the new psychoactive substances EAM-2201 and U-47700, were found exclusively in larvae and hair. The benzodiazepine etizolam was detected only in liver, lungs, and stomach contents, possibly resulting from characteristic tissue distribution in humans and/or larvae., Conclusions: Antemortem external hair contamination with synthetic cannabinoids from side-stream smoke and postmortem hair contamination with substances in putrefaction fluids can be supposed in these cases. Our findings suggest that supplementary information can indeed be gained from analyzing larvae additional to those human specimens that are typically used for toxicological analysis after extensive postmortem decomposition. Nevertheless, these results represent merely two cases, requiring in-depth studies to determine whether such findings can identify acute intoxications as possible causes of death., (© 2021. The Author(s).)

Background: There is increasing evidence for oxytocin as a neurotransmitter in spinal nociceptive processes. Hypothalamic oxytocinergic neurons project to the spinal dorsal horn, where they activate GABA-ergic inhibitory interneurons. The present study tested whether the long-acting oxytocin-analogue carbetocin has anti-nociceptive effects in multi-modal experimental pain in humans., Methods: Twenty-five male volunteers received carbetocin 100 mcg and placebo (0.9% NaCl) on two different sessions in a randomized, double-blinded, cross-over design. Multi-modal quantitative sensory testing (QST) including a model of capsaicin-induced hyperalgesia and allodynia were performed at baseline and at 10, 60 and 120 min after drug administration. QST data were analysed using mixed linear and logistic regression models. Carbetocin plasma concentrations and oxytocin receptor genotypes were quantified and assessed in an exploratory fashion., Results: An anti-nociceptive effect of carbetocin was observed on intramuscular electrical temporal summation (estimated difference: 1.26 mA, 95% CI 1.01 to 1.56 mA, p = .04) and single-stimulus electrical pain thresholds (estimated difference: 1.21 mA, 95% CI 1.0 to 1.47 mA, p = .05). Furthermore, the area of capsaicin-induced allodynia was reduced after carbetocin compared to placebo (estimated difference: -6.5 cm 2 , 95% CI -9.8 to -3.2 cm 2 , p < .001)., Conclusions: This study provides evidence of an anti-nociceptive effect of carbetocin on experimental pain in humans., Significance: This study provides evidence of the anti-nociceptive effect of intravenous administration of the oxytocin agonist carbetocin in healthy male volunteers., (© 2021 European Pain Federation - EFIC®.)

One of the major challenges of testing drugs of abuse is the detection of highly diluted urine samples. The ingestion of a large amount of fluid can considerably reduce the concentration of substances, possibly resulting in inaccurate drug testing. For detection, determination of urinary creatinine is a widely established procedure. In this study, results from the most popular methods, including photospectrometry (Jaffe) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been compared regarding 327 urine abstinence control samples. Because samples with creatinine concentrations close to the cutoff of 20 mg/dL are of particular interest, only samples below 50 mg/dL were considered. Results revealed a close correlation of creatinine concentrations by both analytical methods with an R 2 value of 0.9005. A mean concentration difference of 3.30 ± 3.45 mg/dL was observed, indicating a moderate underestimation by the Jaffe reaction. Graphical analyses showed high accordance between both methods with only a few outliers. Due to easy handling and for economic reasons, the spectrometric method is often preferred over LC-MS/MS. For urine samples with creatinine concentrations close to the cutoff, confirmation through a second method should be performed to avoid a possible disadvantage or even severe consequences for the respective individual. It is recommended that each laboratory establishes a reliable verification method., (© 2021 John Wiley & Sons, Ltd.)

Hair is the matrix of choice in forensic toxicology when retrospective analysis is needed. Nonetheless, due to misalignment, different growth stages and segmentation lengths of 0.5-1 cm, resolution of time is limited. By segmental analysis of single hairs, most of these factors can be compensated and resolution of time is enhanced. A method for manually segmenting single hairs in 2-mm sections and screening for 156 analytes by liquid chromatography coupled to tandem mass spectrometry has been developed and validated. The method was applied to 15 single-dose cases concerning different pharmaceuticals by analyzing 10 hairs each, sampled 1 and 2 months after ingestion in most cases. The validation showed a lower limit of quantification of ≤1.25 pg/segment for ~90% of analytes and good accuracy. Many substances could be detected in the presented cases, whereas detection of benzodiazepines and low dosed opioids remains challenging. In positive cases, characteristic peak-shaped concentration profiles across the hairs were obtained. The segment with most coinciding peak maxima can be allocated to the time of ingestion. A method for the determination of individual hair growth rate was applied and revealed a gap between expected and actual position of peak maxima. Additionally, different localization of simultaneously administered substances was observed. These findings were tried to be explained by different routes of incorporation and may contribute to current knowledge. The presented method may directly be applied to similar questions in hair analysis, and the findings are considered important for interpreting further results in single hair analysis., (© 2021 John Wiley & Sons, Ltd.)

Major studies demonstrating the inhibition of myopia in children and juveniles by low-dose atropine eye drops provide little information on the manufacturing process and the exact composition of the atropine dilutions. However, corneal penetration might significantly vary depending on preservatives, such as benzalkonium chloride (BAC), and the atropine concentration. Since there is a trade-off between side effects, stability, and optimal effects of atropine on myopia, it is important to gain better knowledge about intraocular atropine concentrations. We performed an ex vivo study to determine corneal penetration for different formulations. Atropine drops (0.01%) of different formulations were obtained from pharmacies and applied to the cornea of freshly enucleated pig eyes. After 10 min, a sample of aqueous humor was taken and atropine concentrations were determined after liquid-liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The variability that originated from variations in applied drop size exceeded the differences between preserved and preservative-free formulations. The atropine concentration in the anterior chamber measured after 10 min was only 3.8 × 10 -8 of its concentration in the applied eye drops, corresponding to 502.4 pM. Obviously, the preservative did not facilitate corneal penetration, at least ex vivo. In the aqueous humor of children's eyes, similar concentrations, including higher variability, may be expected in the lower therapeutic window of pharmacodynamic action.

Background : A 22-year-old male with a known history of drug abuse presented to our department with prolonged agitated delirium, myocloni, tachycardia and subfebrile temperature after the deliberate ingestion of opium poppy tea ( Papaver somniferum L.) together with the methaqualone analog SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4( 3H )-quinazolinone) which is sold online as a designer drug. Methods : SL-164 and its hydroxy metabolites were detected in serum and urine via liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Results : The pronounced delirium was treated with benzodiazepines and neuroleptics; temporary medical restraint had to be applied. Symptoms completely resolved over the next 72 h and the patient was discharged on day three able to give consent. Conclusions : Although methaqualone was a popular and widespread sedative in the 1950s and 60 s before its discontinuation in the USA in 1985, derivatives of the methaqualone class have not previously played a large role as drugs of abuse in the rapidly growing market of new psychoactive substances. To our knowledge, this is the first case of agitated delirium with detection of SL-164 and hydroxylated metabolites in a patient's serum and urine.

Driving under the influence of drugs (DUID) is a serious global problem and poses a public health risk. With new psychoactive substances (NPS) entering the illicit drug market several years ago, a significant number of highly potent and harmful drugs have become easily available and the use of these substances may impair a person's ability to drive a vehicle safely. Since NPS are not usually covered in routine toxicological analyses used in DUID investigations, only little is known about their prevalence. To gather more information on the prevalence of NPS in cases of impaired driving, a retrospective study was conducted to determine the prevalence of these drugs in blood samples of DUID suspects in southern Germany. A total of 837 blood samples, which were collected in the German federal states Baden-Württemberg and Bavaria in 2017 and 2018, were reanalyzed for designer stimulants and synthetic cannabinoids by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). For the analysis of synthetic cannabinoids, a more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method was additionally used. A total of 14 cases (1.6%) tested positive for NPS. Designer stimulants were detected in two cases (0.2%) and synthetic cannabinoids were found in 12 cases (1.4%). The rather low prevalence rate of 1.6% estimated in this study suggests that driving under the influence of NPS does not play a large role in southern Germany. Nonetheless, in all cases in which the psychophysical impairment cannot be explained by routine toxicological findings, a screening for NPS should additionally be performed., (© 2020 John Wiley & Sons, Ltd.)

Gilles de la Tourette syndrome (TS) is a complex neurodevelopmental disorder characterized by the presence of motor and vocal tics as well as psychiatric comorbidities such as attention-deficit/hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), depression, and anxiety. The underlying cause of the disease is still unknown, but several lines of evidence suggest a paramount role of the dopaminergic system. Based on the clinical observation that cannabis-based medicine including cannabis and delta-9-tetrahydrocannabinol (THC, dronabinol) may improve TS, alternatively, an involvement of the endocannabinoid system (ECS) has been suggested. In this study we measured cerebrospinal fluid (CSF) levels of the two most important endocannabinoids "N"-arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG), the endocannabinoid-like molecule palmitoyl ethanolamide (PEA), and the lipid arachidonic acid (AA) in a sample of adult patients with TS (n = 20) compared with controls (n = 19) using liquid-liquid lipid extraction and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM). CSF levels of AEA (p = 0.0018), 2-AG (p = 0.0003), PEA (p = 0.02), and AA (p < 0.0001) were significantly increased in TS compared with controls. Levels of 2-AG correlated with the severity of comorbid ADHD (p < 0.01). This is the first study, demonstrating alterations in the ECS suggesting an involvement of this system in the pathophysiology of TS. It can be speculated that elevated endocannabinoid levels either represent secondary changes in order to compensate for alterations in other neurotransmitter systems such as the dopaminergic system, are simply an epiphenomenon or, alternatively, represent the primary cause of TS.

Drug testing in hair can complement conventional blood and urine analysis as it enlarges the window of detection and may allow a differentiation of heavy from moderate or rare use. Databases of drug concentrations in biological matrices are a valuable support in interpreting analytical results. In forensic toxicology, several databases exist especially for blood/serum samples. In the present paper, the concentration distributions of more than 100 legal and illegal drugs such as narcotic drugs, opioids, antidepressants, antipsychotics, benzodiazepines, and major metabolites detected in authentic Caucasian hair samples in our laboratory are summarized. Depending on availability, the proximal sections of 1-6 cm in length were analyzed by liquid chromatography/tandem mass spectrometry following extraction of the finely chopped specimens by ultrasound in methanol. The data may present a helpful basis also for other laboratories for an initial evaluation of their results. However, these statistical data should not be used uncritically without including the circumstances of the particular case and the analytical procedures used. In addition, each laboratory in charge of interpreting results from hair analysis should balance own results as far as available with this data base.

This retrospective report focuses on the diagnosis of the Paxillus syndrome, based on two fatal cases of haemolysis following the consumption of Paxillus involutus . These mushrooms are still consumed regularly, despite earlier reports of life-threatening autoimmune haemolytic anaemia. Such cases are nevertheless rare, and thus far no toxin could be identified that causes this unusual form of mushroom poisoning. All these factors contribute to the difficulty in diagnosing the Paxillus syndrome. The following aspects support the diagnosis in the two cases presented here: Both patients consumed the mushroom oftentimes before, yet allegedly without ill effects. Symptoms occurred 2-3 h after the last consumption, exacerbating into circulatory collapse, multiorgan failure, and death. Disseminated intravascular coagulation was identified as cause of death by autopsy of patient 1. Patient 2 died of multiorgan failure, mainly hepatic. Our mycological analyses could identify the consumed mushroom in both cases as Paxillus involutus . Furthermore, we could exclude anticoagulants and several other drugs as trigger for the haemolysis by post-mortem toxicological analysis. However, findings in each of the two cases may have led to the haemolysis, independent of the consumption of Paxillus involutus . Patient 1 carried the anti-erythrocytic antibody, auto-anti-e. Patient 2 contracted chronic hepatitis C years prior to the current incident. Considering the rarity of the Paxillus syndrome, our findings suggest that these patients were particularly susceptible for haemolysis after consuming this mushroom over a prolonged period. Occurrence of the Paxillus syndrome may thus be restricted to regular consumers of Paxillus involutus mushrooms with an existing predisposition for haemolysis.

5F-ADB is an indazole-based synthetic cannabinoid. In recent years, it has been detected in legal high products as well as in biological samples and is associated with serious adverse health, behavioral effects and even death. Due to the fast pace of the market of synthetic cannabinoids, data on such newly appearing substances are scarce. As pharmacological properties are often investigated in vitro or by using animal experiments, reports on synthetic cannabinoid findings in human samples along with corresponding case history descriptions are valuable for the interpretation of upcoming routine cases. Herein we report five cases with verified 5F-ADB consumption, including three fatalities, a case of driving under the influence of drugs as well as a case of grievous bodily harm. In four cases, 5F-ADB could be detected in blood or plasma. Concentrations were in the range of 0.11-0.57 μg/L. In one instance 5F-ADB consumption was verified by the detection of 5F-ADB metabolites in postmortem body fluids. The described cases illustrate various adverse effects including confusion (possibly even psychosis), collapse, loss of consciousness, unsafe driving style or changing moods that might be attributed to 5F-ADB., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Most recently, the synthetic opioid U-47700 has emerged on the illicit drug market and is sold on the Internet as a "research chemical". Its structure is closely related to the synthetic opioid AH-7921. U-47700 is a μ-opioid receptor agonist with a potency of approximately 7.5 times that of morphine. In this study, postmortem concentrations of U-47700 are presented in 26 fatalities which occurred between April 2016 and August 2017 in the southern part of Germany. In 18 of these cases, quantitative analyses of U-47700 were carried out in femoral blood, heart blood, liver, urine, vitreous humor, pericardial fluid, and gastric content. In five cases, concentrations of U-47700 were determined in femoral blood, whereas in one case, the concentration of U-47700 was analyzed in heart blood. Due to advanced putrefaction, the analysis of U-47700 could only be performed in putrefaction fluid in two cases. Quantification of U-47700 was carried out using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) with electrospray ionization operated in positive mode. The median femoral blood concentration of U-47700 (n = 23) was 610 ng/mL (range: 27-2200 ng/mL). Except for one female, all decedents were male and aged between 23 and 56 years (mean age: 34 years). In all fatalities, the cause of death was attributed to an intoxication with U-47700 either alone or in combination with other psychoactive substances. In 15 of the 26 cases, there was a combined use of U-47700 with other new psychoactive substances (NPS). Therefore, not only new synthetic opioids but also additional NPS including synthetic cannabinoids, new stimulant drugs, and designer benzodiazepines should be included in the routine toxicological screening methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Highly diluted urine is among the most commonly observed factors affecting the validity of urine testing for drug abuse. A minimum creatinine concentration of 20 mg/dL urine has been proposed as a marker for dilution of a urine sample. This study investigates the effect of water consumption on creatinine concentration, as well as its effect on specific gravity and osmolality. In this study, 22 subjects (17 women and 5 men) were included to determine the influence of sex and weight on the impact of excessive water consumption on these markers of urine dilution. The subjects consumed 0.5 L, 1.0 L, and 1.5 L of water, respectively, within 15 minutes. The mean minimum creatinine concentrations (Jaffé reaction) for the void 2 hours after fluid intake were 60.4 mg/dL, 15.8 mg/dL, and 10.9 mg/dL for the respective ingested volumes of water. Mean creatinine concentrations excreted by men were significantly higher than those excreted by women. Participants with a weight below 60 kg tended to excrete lower urine creatinine concentrations. 50% of the volunteers with a BMI < 20 kg/m 2 and 20% of the volunteers with a BMI > 20 kg/m 2 exhibited creatinine concentrations below the threshold value of 20 mg/dL. A similar pattern was established for gravity and osmolality. Due to its simple determination, creatinine may be preferred over specific gravity or osmolality. In order to evaluate the internal dilution of a urine sample for legally defensible drug testing, it may be necessary to account for sex and body weight., (© 2018 John Wiley & Sons, Ltd.)

A 100 μg/L or higher concentration of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood serum is generally assumed to be associated with regular and/or heavy use of cannabis. At present, determination of the extent of cannabis use by means of the concentration of THC-COOH in hair has not been assessed. Therefore, we aimed at establishing a threshold for THC-COOH concentrations in hair to prove frequent consumption by comparing THC-COOH concentrations in 129 corresponding serum and hair samples, respectively. The concentration of THC-COOH in the serum was analyzed by gas chromatography-mass spectrometry and in the hair by liquid chromatography-tandem mass spectrometry. Data were statistically evaluated using receiver operating characteristic curves and contingency tables. Our results suggest that a THC-COOH concentration of ≥4.2 pg/mg in hair was always accompanied by a THC-COOH concentration of at least 100 μg/L in blood serum. Should this be confirmed by further studies of a larger study population, a hair concentration of 4.2 pg/mg THC-COOH can be set as a threshold to predict regular and/or heavy consumption of cannabis even if no corresponding blood sample is available for analysis., (© 2018 John Wiley & Sons, Ltd.)

Fluoride is a common stabilizing agent in forensic toxicology to avoid the frequent problem of degradation of drugs in blood samples especially described for cocaine. In cases only samples with addition of fluoride are available, it is a crucial question if also concentrations of common drugs other than cocaine (amphetamines, opiates and cannabinoids) are affected by fluoride. So far, there are only rare literature data available on discrepant results especially for Δ 9 -tetrahydrocannabinol (THC). In this study, comparative analysis of positive tested paired routine plasma/serum samples (n = 375), collected at the same time point (one device with and one without fluoride), was carried out with special focus on cannabinoids. Samples were measured with validated routine liquid chromatography-tandem mass spectrometry methods for THC, 11-hydroxy-THC (THC-OH), 11-nor-9-carboxy-THC (THC-COOH), cocaine, benzoylecgonine, ecgonine methyl ester, morphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxyethylamphetamine, and results were statistically evaluated. Beside the expected stabilization effect on cocaine and the consequently reduced concentration of ecgonine methyl ester in fluoride samples, benzoylecgonine was elevated compared to respective samples without fluoride. Most importantly, new findings were significantly reduced mean concentrations of THC (- 17%), THC-OH (- 17%), and THC-COOH (- 22%) in fluoride samples. Mean amphetamine concentration was significantly higher in samples with the additive (+ 6%). For the other amphetamine type of drugs as well as for morphine and codeine, no significant differences could be seen. Whenever specified thresholds have been set, such as in most European countries, the use of different blood sample systems may result in a motorist being differently charged or prosecuted. The findings will support forensic toxicologists at the interpretation of results derived from fluoride-stabilized blood samples.

Testing hair for cannabis use has increasingly been scrutinised due to exposure to second-hand smoke or environmental contamination. Confirmation of drug use involving detection of metabolites such as 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) having very rarely been considered. We developed a new, simplified procedure with regard to expenditure of time and material to determine delta-9-tetrahydrocannabinol (THC, qualitatively), as well as THC-OH and THC-COOH (quantitatively) from 587 hair samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was compared to hitherto established methods (n = 3). Compared to conventional methanolic extraction alkaline dissolution resulted in higher concentrations for THC-OH. Concentrations determined from specimens ranged from 0.01 to 18.7 ng THC/mg hair, 0.05-37.6 pg THC-OH/mg hair, and from 0.1 to 54.3 pg THC-COOH/mg hair. THC was detectable in 70.4% samples along with both metabolites from more than half of these samples. In 12.9% of THC-positive cases, neither THC-OH nor THC-COOH were present. In 8.9% of THC-negative cases, it was possible to detect metabolites either alone or in combination. THC-OH could more frequently be detected than THC-COOH and appeared to be less susceptible to cosmetic treatment. In summary, THC-OH turned out to be a further suitable marker to prove cannabis use. Determination of both metabolites is recommended to unequivocally differentiate consumption from external exposure or contamination., (© 2018 John Wiley & Sons, Ltd.)

Since there are no characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma need to be confirmed by suitable biomarkers. The postmortem identification of preexisting hyperglycemia or diabetic coma can be difficult if the matrices for the determination of the established biomarkers are not available or the obtained results are close to the established cut-off values. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. To evaluate postmortem 1,5-AG concentrations in vitreous humor (VH) and cerebrospinal fluid (CSF), a liquid chromatographic mass spectrometric method for the quantification of 1,5-AG in VH and CSF was developed and validated according to international guidelines. In order to establish a cut-off for the identification of an ante mortem existing diabetes and the diagnosis of a diabetic coma in deceased the relationships between 1,5-AG concentrations in VH and CSF to other diabetes associated biochemical parameters of 47 non-diabetic, 86 diabetic and 9 cases of diabetic coma were examined. In 83 of these cases, both matrices could be obtained and analyzed. Comparisons of the respective HbA1c, Glucose in VH or Sum-formula of Traub to 1,5-AG concentrations in VH and CSF resulted in correlation coefficients R 2 ≤0.2. For the application of 1,5-AG concentrations in VH against CSF, a linear regression gave a correlation coefficient of R 2 =0.955. Comparable linear correlations of 1,5-AG concentrations could be observed between VH and femoral venous blood (FVB) (R 2 =0.839) as well as between CSF and FVB (R 2 =0.756). Due to overlapping concentration ranges, the determination of a reliable cut-off for the differentiation of diabetic disease to diabetic coma cases was not possible. However, the 1,5-AG concentrations in VH and CSF in cases of deceased diabetics were significantly lower (p<0.05) than in non-diabetic deceased and therefore indicate a pre-existing diabetes or even a diabetic coma as the cause of death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Drugs which are commonly smoked or sniffed (e.g. cocaine), can contaminate hair through smoke or dust; therefore testing for metabolites, especially hydroxy metabolites, is highly recommended. The presence of hydroxy metabolites in street-cocaine (COC) has been discussed. To check if detection of hydroxy metabolites definitely proves ingestion, the presence of these metabolites in street COC samples has to be checked. It is expected that the more hydrophilic hydroxy metabolites of COC are incorporated into the hair-matrix to a lesser extent. For this study 576 COC positive hair samples (≥0.1ng COC/mg hair) were analysed by LC-MS/MS for benzoylecgonine (BE), norcocaine (NC), cocaethylene (CE), ortho-, meta- and para-hydroxy COC (o-, m-, p-OH-COC), meta- and para-hydroxy BE (m-, p-OH-BE), and meta- and para-hydroxy NC (m-, p-OH-NC). The results were compared with the respective metabolite/COC concentration ratios in 146 street COC samples, confiscated by the Bavarian police. Peak areas were used to estimate BE/COC, NC/COC, CE/COC and hydroxy metabolites/COC. Similar metabolic ratios were found for o-OH-COC in 88% of the samples, but for p-OH-COC and m-OH-COC only in 5.1% and 6.8%, respectively. Notably, p- and m-OH-BE as well as p- and m-OH-NC could not be identified from seized samples. We propose that area ratios exceeding the ratios of street COC more than twice or identification of OH-BE and OH-NC enable to differentiate COC consumption from contamination. Using these criteria, consumption of the drug could be proven in 92% of COC positive samples. As detection of meta- and para-hydroxy metabolites using the above mentioned criteria is a reliable tool to distinguish between ingestion and external contamination, it is recommended to implement their measurement into daily routine work., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Because of the lack of characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma can be complicated. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. Next to clinical applications determination of 1,5-AG can also be used in forensic analysis. To investigate the elimination of 1,5-AG, a liquid chromatographic-mass spectrometric method for the determination of 1,5-AG and creatinine in urine was developed and validated according to international guidelines. To evaluate ante mortem concentrations of 1,5-AG spot urine samples of 30 healthy subjects, 46 type 1 and 46 type 2 diabetic patients were analyzed. 1,5-AG urine concentrations of diabetic patients were significantly (p<0.001) lower (mean: 1.54μg/ml, n=92) compared to concentrations of healthy subjects (mean: 4.76μg/ml, n=30) which led to the idea that 1,5-AG urine concentrations post mortem might help in the interpretation of a diabetic coma post mortem. Urine of 47 deceased non-diabetics, 37 deceased diabetic and 9 cases of diabetic coma were measured. Comparison of blood and urine 1,5-AG concentrations in clinic samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation. Urinary levels of 1,5-AG in deceased diabetic (mean 6.9μg/ml) and in non-diabetic patients (mean 6.3μg/ml) did not show a significant difference (p=0.752). However, urinary 1,5-AG concentrations in deceased due to diabetic coma (mean: 1.7μg/ml) were significantly lower than in non-diabetic (mean: 6.3μg/ml, p=0.039) and lower than in diabetic cases (mean: 4.7μg/ml, p=0.058). The determination of a reliable cut-off for the differentiation of diabetic to diabetic coma cases was not possible. Normalization of urinary 1,5-AG concentrations with the respective creatinine concentrations did not show any gain of information. In clinical (serum) and forensic blood samples a significant difference between all groups could be detected (p<0.05). Comparison of blood and urine 1,5-AG concentrations in clinical samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC-positive samples for these compounds was performed by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Once identified, mass transitions for o-, p- and m-isomers of hydroxy COC as well as p- and m-isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC-positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p-OH-BE, m-OH-BE as well as p-OH-NCOC and m-OH-NCOC have been identified for the first time in COC-positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC-positive hair samples., (Copyright © 2017 John Wiley & Sons, Ltd.)

In this study, two fatalities associated with the synthetic opioids AH-7921 and MT-45 are reported. Within the last few years, both compounds have emerged on the recreational drug market and are sold as "research chemicals" on the internet. In the first case, a 22-year-old woman was found dead in the bedroom of her apartment by two of her friends. A plastic bag labeled "AH-7921" was found in the apartment and the two friends stated that the deceased had consumed AH-7921 prior to her death. The woman was a known drug addict. In the second case, a 24-year-old man was found dead in his room by his mother. The deceased was sitting on a chair in front of his desk slumped over. Several bags of white powder labeled "MT-45", "Methoxmetamine" and "Methoxphenidine" were found in his room. Toxicological analyses of femoral blood, heart blood, liver, pericardial fluid, urine, vitreous humor and stomach content of the deceased were performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Time-of-flight mass spectrometry was carried out on an LC-Triple TOF 5600 system (AB Sciex) with electrospray ionization operated in positive mode. In the first case, additional hair analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-QTOF-MS. In both cases, the relevant synthetic opioid could be detected in all analyzed samples. The concentration of AH-7921 was determined to be 450μg/L in femoral blood. MT-45 was present at a concentration of 2900μg/L in femoral blood. Besides methoxmetamine which could qualitatively be detected in femoral blood, urine and stomach content no methoxphenidine was found. In summary, deaths of the young individuals could be, by exclusion of other causes of death, attributed to the consumption of an overdose of AH-7921 and MT-45, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This work represents the development, validation, and application of a liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) screening method for the detection of pharmaceutical substances and illicit drugs (acidic, basic, and neutral organic drugs) in urine samples. Time-of-flight mass spectrometry was performed using an LC-Triple TOF 5600 system with electrospray ionization operated in both positive and negative mode, respectively. The limits of detection (LODs), determined for 34 substances, were < 10 ng/mL for 91% of the compounds. The limits of quantitation (LOQs) were < 20 ng/mL for 91% of the substances. The identification of the compounds was based on exact mass (< ± 5 ppm), retention time (<2%) if available, isotopic pattern fit (<10%) and library hit (>70%). These four parameters served as identification criteria and are discussed according to their role in identifying compounds even without reference substances. In routine casework, two in-house XIC (extracted ion chromatogram) lists, consisting of 456 protonated and 26 deprotonated compounds were used and retention times for 365 compounds were available. Compared to the results found with the established gas chromatography-mass spectrometry (GC-MS) procedure, the findings with the LC-QTOF-MS screening method showed a good comparability. Results that were not detected by LC-QTOF-MS because of a missing entry in the targeted XIC list could retrospectively be confirmed by simply entering the elemental formula of the relevant substance into the software and reprocessing the sample. LC-QTOF-MS offers an attractive technique for the fast and specific identification of illicit drugs and toxic compounds in urine samples. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Discrimination between street heroin consumption and poppy seed ingestion represents a major toxicological challenge in daily routine work. Several difficulties associated with conventional street heroin markers originate from their versatile occurrence in various poppy seed products and medications, respectively, as well as to small windows of detection. A novel opportunity to overcome these hindrances is represented by the new potential street heroin marker acetylated-thebaine-4-metabolite glucuronide (ATM4G), originating from thebaine during street heroin synthesis followed by metabolic reactions after administration. In this study, urine samples after consumption of different German poppy seed products and urine samples from subjects with suspicion of preceding heroin consumption were tested for ATM4G, 6-AC (6-acetylcodeine), papaverine, noscapine, 6-MAM (6-monoacetylmorphine), morphine, and codeine. Neither 6-AC and 6-MAM nor ATM4G but morphine and codeine could be detected in urine samples following poppy seed ingestion. As well, neither papaverine nor noscapine could be observed even after consumption of poppy seeds containing up to 37 µg noscapine and up to 9.8 µg papaverine, respectively. Concerning the urine samples with suspicion of preceding heroin consumption, ATM4G could be detected in 9 of 43 cases. By contrast, evidence of 6-AC and 6-MAM, respectively, could only be seen in 7 urine samples. In conclusion, ATM4G should be measured additionally in cases requiring discrimination of street heroin consumption from poppy seed intake. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Two cases of fatalities are reported of which the recreational use of Mitragyna speciosa ("kratom") could be confirmed. One of these cases presents with one of the highest postmortem mitragynine concentrations published to date. Our results show that even extremely high mitragynine blood concentrations following the consumption of kratom do not necessarily have to be the direct cause of death in such fatalities as a result of an acute overdose. The two cases are compared with regard to the differences in mitragynine concentrations detected and the role of mitragynine in the death of the subjects. Irrespective of the big differences in mitragynine concentrations in the postmortem blood samples, mitragynine was not the primary cause of death in either of the two cases reported here. Additionally, by rough estimation, a significant difference in ratio of mitragynine to its diastereomers in the blood and urine samples between the two cases could be seen., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The Mombello Psychiatric Hospital in Limbiate, near Milan, replaced the old Senavra Hospital as the Psychiatric Hospital for the Province of Milan in the 19th century. During the early 20th century, bodies of several Mombello patients were dissected and preserved by Giuseppe Paravicini, an anatomist who operated within the asylum. The aim of the present study was to examine and memorialize this important assemblage. To this end, we were allowed to sample the head hair of six such preparations for toxicological analysis. By means of high performance liquid chromatography, cocaine and its main metabolite, benzoylecgonine, were detected in two out of six hair samples. The concentrations for cocaine were 0.151 and 0.09ng/mg and for benzoylecgonine 0.103 and 0.147ng/mg, respectively. Given that cocaine was a commonly used medicine, beginning in the mid-19th century and persisting into the 20th century, it is not surprising that some patients may have ingested this drug. In addition to the detection of cocaine, these analyses also provided evidence of nicotine and caffeine intake., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The organic cation transporter OCT1 (SLC22A1) mediates uptake and metabolism of the active tramadol metabolite (+)O-desmethyltramadol in the liver. In this study, the influence of OCT1 genetic polymorphisms on pharmacokinetics and analgesic efficacy of tramadol in patients recovering from surgery was analyzed in addition to the CYP2D6 genotype. Postoperative patients who received tramadol through patient-controlled analgesia were enrolled. Genotypes resulting in 0, 1, or 2 active OCT1 alleles were determined as well as CYP2D6 genotypes. The primary endpoint was the 24-hour postoperative tramadol consumption in patients with 0 vs at least 1 active OCT1 allele. Secondary endpoint was the OCT1-dependent plasma concentration (areas under the concentration-time curves) of the active tramadol metabolite (+)O-desmethyltramadol. Of 205 patients, 19, 82, and 104 carried 0, 1, and 2 active OCT1 alleles, respectively. Cumulative tramadol consumption through patient-controlled analgesia was lowest in patients with 0 active OCT1 allele compared with the group of patients with 1 or 2 active alleles (343 ± 235 vs 484 ± 276 mg; P = 0.03). Multiple regression revealed that the number of active OCT1 alleles (P = 0.014), CYP2D6 (P = 0.001), pain scores (P < 0.001), and the extent of surgery (0.034) had a significant influence on tramadol consumption. Plasma areas under the concentration-time curves of (+)O-desmethyltramadol were 111.8 (95% confidence interval: 63.4-160.1), 80.2 (65.1-95.3), and 64.5 (51.9-77.2) h·ng·mL in carriers of 0, 1, or 2 active OCT1 alleles (P = 0.03). Loss of OCT1 function resulted in reduced tramadol consumption and increased plasma concentrations of (+)O-desmethyltramadol in patients recovering from surgery. Therefore, analyzing OCT1 next to CYP2D6 genotype might further improve future genotype-dependent dose recommendations for tramadol.

Synthetic amphetamines such as 3,4-methylenedioxy-N-methylamphetamine (MDMA, Ecstasy) have become recreational drugs in German discotheques because of their euphoric and mood-brightening effects. However, their consumption involves considerable risks, which may even be lethal under certain circumstances. A 19-year-old man was taken to a university hospital for suspected intoxication with a narcotic drug, where he died the next day. As cause of death "fulminant liver failure" was diagnosed. In blood from the femoral vein, MDMA was found in a concentration of 4.27 mg/l. Histological examination showed acute necrosis of the liver and parenchymatous bleeding. The second case is that of a 39-year-old man who collapsed at his workplace and died in hospital shortly afterwards. In his rucksack, a small bag with 1.6 g of amphetamine was found. Analysis of blood from the femoral vein showed an amphetamine concentration of 1.08 mg/l.

Fatty acid ethyl esters (FAEE), direct metabolites of ethanol, are suitable alcohol markers that can be detected in different tissues. The determination of FAEE in hair can help to evaluate social and excessive alcohol consumption. Due to the presence of FAEE in the hair of teetotalers, proving alcohol abstinence seems to be impossible. To verify these results, an solid phase micro extraction-gas chromatography/mass spectrometry procedure for the determination of the four FAEE: ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate in hair was validated with special focus on low concentration levels. Besides very high sensitivity (limits of detection between 0.005 and 0.009 ng/mg), good results for linearity, precision and accuracy, recovery and stability were achieved. In addition, 73 hair samples with measured ethyl glucuronide (EtG) concentrations between 4 and 10 pg/mg were analyzed for FAEE. By using the following cut-offs: EtG: 7 pg/mg, FAEE: 0.2 ng/mg a satisfying matching rate of 72.6% was found. This shows that FAEE can be determined to verify borderline EtG concentrations even in the context of abstinence tests. However, the diversified influencing factors on analyte concentrations in hair, which may explain the large deviations between EtG and FAEE results observed in some cases, have to be mentioned when interpret ambiguous results., (© The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Levamisole is an anti-helminthic drug and gained forensic interest after it was found that it was used as a cocaine adulterant. A liquid chromatography-mass spectrometry (LC-MS) method for the determination of levamisole and its metabolite aminorex in human plasma is described. Selectivity is given; calibration curves were linear within a calibration range of 1 ng/mL-500 ng/mL. Limits of detection and quantification (LODs, LOQs) were 0.85 ng/mL for levamisole and 0.09 ng/mL, and 0.34 ng/mL for aminorex, respectively. Precision data was in accordance with the GTFCh guidelines. The validated method was successfully applied to study the pharmacokinetics of levamisole after administration of 100 mg of levamisole orally. Levamisole could be detected up to 36 h after ingestion in serum, while aminorex never exceeded the LOQ. A one-compartment model best described levamisole pharmacokinetics. The following parameters were calculated: ka = 1.2 [1/h], CL/F = 52 l/h, V/F = 347 l, f (renal) = 0.0005, t ½ = 2.0 h, AUC = 1923 ng/mL*h, cmax = 214 ng/mL, tmax = 1.98 h. Levamisole could be quantified in 42.5% of cocaine--positive plasma samples (2.2 to 224 ng/mL). Aminorex was positive in only 11.3% of the cases; however, it was never found higher than the LOQ. Pemoline, another stimulant detected in horse urine samples after administration of levamisole, was not found either in serum or in urine of this pharmacokinetic study. In post-mortem cases, levamisole and aminorex could be detected in femoral blood and the urine of cocaine users. Pemoline was not detected., (Copyright © 2014 John Wiley & Sons, Ltd.)

A massive lethal overdose with beta-metildigoxin in a 36-week-old infant is presented. Determination of beta-metildigoxin and its metabolites digoxin, digoxigenin and digoxigenin-monodigitoxosid is achieved by a liquid chromatographic mass spectrometric (LC-MS/MS) method. Measured concentrations for beta-metildigoxin and digoxin in peripheral blood were 40.2 ng/ml and 25.6 ng/ml, respectively. Tissue distribution showed highest concentrations in kidney tissue and gastric content. The metabolite digoxigenin-monodigitoxosid could be detected in heart blood, duodenal content, gastric content and fat tissue while the metabolite digoxigenin could only be detected in gastric content since the drug was given by a stomach tube., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

There is a need for quick and reliable methods for rapid screening of drug-influenced drivers on the roadside by police. Because the window of detection in oral fluid is more similar to blood than to urine, this matrix should therefore be appropriate for screening procedures. The performance of the Rapid STAT(®) (Mavand Solution GmbH, Mössingen, Germany), DrugWipe5/5+(®) (Securetec Detektions-Systeme AG, Brunnthal, Germany) and Dräger DrugTest(®) 5000 (Draeger Safety AG & Co. KGaA, Luebeck, Germany) on-site oral fluid devices was evaluated with random oral fluid specimens from car drivers in North Rhine-Westphalia (Germany). Additionally, some drivers were checked using an on-site urine device (DrugScreen(®), NAL von Minden, Regensburg, Germany). During a 11-month period, 1.212 drivers were tested. Both OF and urine on-site tests were compared to serum results. The following sensitivities were obtained by the oral fluid devices: THC 71% (DrugWipe(®)), 87% (Dräger), 91% (RapidSTAT); opiates 95% (Dräger), 100% (DrugWipe(®), RapidSTAT(®)); amphetamine 84% (DrugTest(®) 5000), 90% (RapidSTAT(®)), 100% (DrugTest(®) 5000); methamphetamine 50% (DrugTest(®) 5000), 100% (RapidSTAT(®)); cocaine 76% (DrugTest(®) 5000), 100% (DrugWipe(®), RapidSTAT(®)); methadone 33-63%, and benzodiazepines 0-33% (both with a low number of positives). THC specificity was especially low (29% [DrugWipe(®)] and 47% [DrugTest(®) 5000]) due to low cut-off concentrations. These data were similar to those obtained from the literature (e.g., DRUID project). The urine screening device showed a good sensitivity (THC 93%, opiate 94%, amphetamine 94%, methamphetamine 75% (low number of positives), cocaine 100%) and also an acceptable specificity (39%, 86%, 63%, 77%, 47%, respectively). Although oral fluid may be a useful matrix for on-site testing of drugged drivers, it is evident that oral fluid devices still show a lack of sensitivity (methamphetamine, benzodiazepines) and specificity (THC). Poor results for benzodiazepines may be explained by the small positive test number. Although the sensitivity for THC came out higher than compared to the literature, specificity is not yet satisfactory (only <90%). Furthermore, specificity was poor due to lowered cut-offs resulting in multiple false positive tests., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The imidazoline derivative xylometazoline, an alpha-2-adrenergic agonist, is used as non-prescription nasal preparation due to its vasoconstrictive and decongestive properties. Especially in children, an overdose can quickly cause severe central nervous system depression and cardiovascular adverse effects. In three 3-year-old boys (triplets) a xylometazoline intoxication was diagnosed by toxicological analysis. On admission to an emergency unit all three children were still unresponsive. One triplet showed respiration of 15-20 breaths/min and required oxygen support (3L/min) via face mask; his electrocardiogram revealed sinus bradycardia of 64 beats/min with supraventricular extrasystoles. However, no interventions were necessary except fluid management via intravenous lines. Eleven hours after the event, two of the triplets were awake but still not fully oriented. The third triplet woke up 20h after instillation of nose drops. Intoxication was caused by a compounding error in a pharmacy resulting in a concentration 40 times above the adequate dosage for children. In general, physicians, pharmacists and the public should be educated about the toxicity of over-the-counter preparations., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Recreational use of synthetic cannabinoid receptor agonists-so-called "Spice" products-became very popular during the last few years. Several reports on clinical symptoms and poisonings were published. Unfortunately, most of these reports do not contain any analytical data on synthetic cannabinoids in body fluids, and no or only a limited number of cases were reported concerning driving under the influence (DUI) of this kind of drugs. In this article, several cases of DUI of synthetic cannabinoids (AM-2201, JWH-018, JWH-019, JWH-122, JWH-210, JWH-307, MAM-2201 (JWH-122 5-fluoropentyl derivative), and UR-144) are presented, focusing on analytical results and signs of impairment documented by the police or the physicians who had taken the blood sample from the suspects. Consumption of synthetic cannabinoids can lead to impairment similar to typical performance deficits caused by cannabis use which are not compatible with safe driving. These deficits include centrally sedating effects and impairment of fine motor skills necessary for keeping the vehicle on track. Police as well as forensic toxicologists and other groups should become familiar with the effects of synthetic cannabinoid use, and be aware of the fact that drug users may shift to these "legal" alternatives due to their nondetectability by commonly used drug screening tests based on antibodies. Sophisticated screening procedures covering the complete range of available compounds or their metabolites have to be developed for both blood/serum and urine testing.

The Capuchin Catacombs of Palermo contain over 1800 preserved bodies: friars, priests and laypeople including men, women, and children. The bodies were accessible to family members who could visit the deceased and commemorate them through prayers. The "Sicily Mummy Project" analyzed hair samples from 38 mummies to determine the presence of ethyl glucuronide (EtG) using a routine procedure in our accredited laboratory of liquid chromatography coupled with mass spectrometry. The limit of quantification was 2.3 pg/mg. The hair samples were from 1.5 to 12 cm in length. All samples were analyzed in 2 segments (seg. A 0-3 cm and seg. B the remainder). Samples <4 cm in length were cut in half. In 31 out of 76 segments positive results were obtained for EtG, with concentrations between 2.5 and 531.3 pg/mg (mean 73.8, median 13.3 pg/mg). In 14 cases positive results were obtained for both segments. In one sample a positive result was obtained for segment A but not for segment B and in a further two samples only for segment B. The results indicate that EtG analyses can be performed on mummy hair samples even several hundred years after death to identify evidence for significant alcohol consumption during life., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The appearance of dangerous and insufficiently studied designer drugs has increased substantially within the last few years. Mixtures containing centrally active compounds are often declared as "bath salt", "incense", "plant food", "bong cleaners" and are marketed in head shops and on the Internet. As the majority of the ingredients of such products are not subject to regulations of the German Narcotics Law (Betäubungsmittelgesetz, BtMG), the vendors and consumers mistake the sale of such products for legal. An alternative possibility to prosecute the distribution of so-called "legal highs" arises from the regulations of the German Medicinal Products Act (Arzneimittelgesetz, AMG). Indicating a private address, several products were purchased via the Internet. The products were analyzed by gas chromatography- mass spectrometry using computer-assisted database search and potential hits were checked for plausibility. The analysis of 100 samples revealed centrally acting compounds (including caffeine) in 98 % (75 % of all samples positive for caffeine). In 16 % of the samples, drugs subject to the BtMG at the time of purchase (end of 2011) were found including 2,5-dimethoxy-4-methylamphetamine, amphetamine, etilamphetamine, N-benzylpiperazine, mephedrone, methcathinone, and phenobarbital. In 55 % of the samples, drugs subject to the current BtMG were found (after its amendment on 20 July 2012). In 37 % of the samples, substances subject to the AMG were found (e.g. ephedrine). In 35 % of the samples, drugs with a potential psychotropic effect were found. In 57.3 % of the positive samples, more than one active ingredient was determined and in some cases up to five active components were found. Other interesting pharmacologically active ingredients found were 4-methylcathinone (n=13), flephedrone (n=8), trifluoromethylphenyl-piperazine (n=7), methylone (n=5), butylone (n=2), hordenine (n=2), and harmane (n=2). Most of the substances not covered by the BtMG can be classified as "unsafe" drugs. The distribution of unsafe drugs is illegal in Germany. However, the easy availability of real or potential drugs has to be seen critically. Little is known about the toxicological and pharmacological effects of those single substances, let alone of interactions in mixtures of such substances. In chat rooms, users advertise such drugs and blaze abroad their own experiences.

This article presents a case of suicide by intoxication with various pharmaceuticals, particularly anticonvulsants, combined with the fungicide captan. A cause of death could not be ascertained at autopsy. However, systematic toxicological analysis (STA) including a screening via solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for (semi) volatile organic compounds revealed results suggesting a possible cause of death. The effects of captan on the human organism, its metabolism, and distribution will be discussed. Macroscopically, the cause of death was unascertained. STA revealed clonazepam, citalopram, and its metabolites, lamotrigine, levetiracetam, lacosamide, clonazepam, captan, and its metabolite tetrahydrophthalimide (THPI). For the first time, it was detected in human viscera. A quantification of THPI was performed to obtain distribution in the organs. The significance of a complete STA must be emphasized. The presence of THPI would have been missed without previous detection of captan. Consequently, this fatality would not have been investigated satisfactorily., (© 2013 American Academy of Forensic Sciences.)

A 42-year old-man was found lying in his bed having seizures. Later he became unconscious and hypotonic developing mydriasis as well as rigidity. The body core temperature (rectal temperature) was above 42 °C. Blood pH was decreased during treatment, and his general condition deteriorated. The patient developed gasping respiration, ventricular fibrillation, and died. During autopsy and histological investigation cerebral and pulmonary edema were noted together with general congestion of the internal organs. Further observations included contraction bands of myocytes, a contracted spleen, fibrosis of the liver, and gall stones. Toxicological analyses of peripheral blood revealed the following results: amisulpride 4.65 mg/l, biperiden 0.12 mg/l, imipramine 0.33 mg/l, and desipramine 0.68 mg/l. An amisulpride-induced neuroleptic malignant syndrome was therefore diagnosed as the patho-physiological mechanism leading to death.

In a preliminary test, four single hairs of a drug abuser were analyzed for the presence of drugs by MALDI Fourier transform mass spectrometry (MALDI-FTMS). Washed hair strains were directly fixed on a sample plate using pressure stable, double-sided adhesive tape; α-cyano-4-hydroxycinnamic acid matrix was manually spotted onto the hair strains. FTMS full scans were obtained moving from the hair root region towards the hair tip. Cocaine (accurate m/z ratio 304.15433) was identified mostly from the root of the hair and then later again towards the hair tip. This was confirmed by analysis of a second hair. Additionally cocaine metabolites with m/z ratio 290.13868 (benzoylecgonine), and m/z 318.16998 (cocaethylene) were detected for plausibility control. Using the MALDI technique, time-related information was obtained concerning the behavioural pattern of the consumer with high resolution compared to conventional procedures. However, in two hairs of the same individual which were analyzed under the same conditions, negative results were achieved. These preliminary results confirm the applicability of MALDI-MS for the determination of drugs and pharmaceuticals in hair samples being useful in forensic toxicology. The high chronological resolution allows an enhanced interpretation concerning the periods of drug administration. However, the negative results with two negative hairs have also demonstrated that hair analysis of single hairs can lead to misinterpretation. Different growth rates have to be considered, and particularly the phenomenon of different growth phases (anagen, catagen, telogen) require attention., (Copyright © 2013 John Wiley & Sons, Ltd.)